MECHANISM OF ALLOSTERIC INFLUENCE ON ENZYME ACTIVITY

本申请的长期目标是了解分子生物学。 变构配体改变酶行为的机制。 具体地说，猪心胰蛋白酶，E。杆菌 牛心脏NAD依赖性异柠檬酸氨甲酰磷酸合成酶 脱氢酶和大鼠肝磷酸果糖激酶。 富马酸酶和氨甲酰磷酸合成酶分别由 影响最大速度和/或米氏反应的变构调节剂 通过改变蛋白质的三级结构来改变常数。 动力学 这些酶的行为将研究使用热力学 链接功能方法。 这种方法提供了定量的 修饰配体与游离酶的解离常数， 修饰配体对最大速度的分数影响，以及 底物与变构物质之间的相互作用自由能 配体。 后两个参数描述了 变构配体一旦结合。 通过观察这些参数的变化 通过系统地改变变构配体的结构而产生 或蛋白质，我们将能够推断配体的结构特征， 蛋白质对于变构配体作用的这两个独立方面是重要的。 这种方法将被扩展到调查的作用机制， 也改变酶的聚集状态的变构配体， 将应用于异柠檬酸脱氢酶的调节研究， ADP和磷酸果糖激酶通过多种调节配体。 异柠檬酸脱氢酶应该提供一个简单的例子， 三级和四级变构作用之间的关系， 而磷酸果糖激酶代表了几种 不同的相互依赖的调节机制。 尽管如此复杂， 重要的新的洞察力应该通过定量评估， 相关的自由能耦合参数。 这种洞察力一直被掩盖 在过去的共同假设，酶可以存在于一个 只有两种可能的构象状态 这一新的见解尤其是 重要的是，鉴于磷酸果糖激酶在 碳水化合物体内平衡 这些研究所需的实验方法主要包括 酶动力学和荧光偏振测量，后者 在用合适的外源性共价标记的酶上进行 荧光探针

The long term objective of this application is to understand the molecular mechanisms by which allosteric ligands modify enzymatic behavior. Specifically, the allosteric properties of porcine heart fumarase, E. coli carbamoyl phosphate synthetase, bovine heart NAD-dependent isocitrate dehydrogenase, and rat liver phosphofructokinase will be investigated. Fumarase and carbamoyl phosphate synthetase are each regulated by allosteric modifiers that influence maximal velocity and/or Michaelis constants by altering the tertiary structure of the protein. The kinetic behavior of these enzymes will be studied using a thermodynamic linked-function approach. This approach provides for the quantitation of the dissociation constant of modifying ligand from free enzyme, the fractional influence that the modifying ligand has on maximal velocity, and the free energy of interaction between the substrate and allosteric ligand. These latter two parameters describe the efficacy of the allosteric ligand once bound. By observing the changes in these parameters produced by systematically altering the structure of the allosteric ligand or protein, we will be able to infer structural features of the ligand and protein important for these two separate facets of allosteric ligand action. This approach will be extended to investigate the mechanism of action of allosteric ligands that alter an enzyme's aggregation state as well and will be applied to a study of the regulation of isocitrate dehydrogenase by ADP, and phosphofructokinase by a wide variety of regulatory ligands. Isocitrate dehydrogenase should provide a straightforward example of the relationship between tertiary and quaternary levels of allosteric action, whereas phosphofructokinase represents a complex example of several different interdependent regulatory mechanisms. Despite this complexity, significant new insight should be obtained by quantitatively evaluating the pertinent free energy coupling parameters. This insight has been obscured in the past by the common assumption that the enzyme can exist in one of only two possible conformational states. This new insight is especially important in view of the vital role phosphofructokinase plays in carbohydrate homeostasis. The experimental methodologies required for these studies consist primarily of enzyme kinetics and fluorescence polarization measurements, the latter being performed on enzymes covalently labeled with a suitable extrinsic fluorescent probe.